The path by which eucaryotic large ribosomal RNAs are produced is well defined, but little is known about the specific reactions. The present application proposes to characterize the role of specific small nuclear RNAs (snRNAs) required for rRNA processing in Saccharomyces cerevisiae. The snRNAs will be investigated in two contexts, involving: a) extension of ongoing studies of a species known as U14 and, b) assessing the roles of yet-uncharacterized snRNAs associated with the primary processing complex. U14 is phylogenetically conserved and required for production of 18S rRNA; its loss results in incorrect processing of the 35S rRNA transcript and rapid turnover of abnormal intermediates containing 18S RNA. In vivo cross-linking data show that U14 interacts directly with precursor rRNA, through an essential complementary sequence. Several other essential elements of U14 have been defined and a secondary structure map is available. Goals for the project period include: 1) defining the interaction of U14 with pre-rRNA, 2) establishing a cell-free assay of U14 function, 3) characterizing proteins of the U14 snRNP, 4) describing the synthesis of U14 itself, and 5) characterizing novel snRNAs of the 90S rRNA processing complex. Interaction of U14 and 18S RNAs will be examined by mutation of sequences required for in vivo cross-linking and processing function. Efforts to establish a cell-free assay of U14 function will focus on the pre-rRNA cleavage reaction 5' proximal to 18S RNA (A1 site), using natural and in vitro produced rRNA substrates and extracts depleted of U14. Proteins of the U14 snRNP will be identified by characterizing genes that suppress defects in U14 function. Protein binding sites on U14 will be identified biochemically and the role of the proteins in snRNP formation and rRNA processing will be analyzed genetically and immunologically. DNA signals involved in U14 transcription, processing and regulation will be defined by mutagenic analysis. Finally, the snRNAs associated with the processing complex will be catalogued and the roles of new species assessed by in vivo depletion analysis. Results from the proposed studies will yield important insights into the role of the snRNAs in eucaryotic cells and establish general approaches for characterizing these vital RNAs in more complex organisms, including humans.